In conventional chromatography, separation between analytes is achieved due to migration of the analytes through a chromatography column at different speeds. The difference in speed is a result of differences among the analytes with regard to their rates of adsorption to and desorption from the stationary phase of the column. A problem encountered with conventional chromatography is that the width of an analyte band increases as the analyte migrates along the column, leading to elution of the analyte in a diluted form. Elution in a concentrated form (as a narrow peak) requires the sample containing the analyte to be injected in a small volume, i.e. the sample should be concentrated. Frequently, a concentrated sample is not available, and an additional step of concentrating the sample is needed to prevent elution in a diluted form. In addition, an analyte peak is often distorted in shape due to factors such as dead volume, unwanted analyte-surface interactions, and injection errors.
Focusing of an analyte band to yield narrow analyte peaks has been achieved in a form of gas chromatography known as thermal gradient gas chromatography (TGGC) in which a spatial temperature gradient is established along the column axis to achieve focusing. In a typical TGGC, temperature decreases from the column inlet to the column outlet causing the front of the analyte peak to experience a lower temperature, and therefore, migrate at a lower speed compared to the rear of the peak, thereby resulting in a focusing effect. In contrast, in conventional gas chromatography, i.e., in the absence of a spatial thermal gradient, analyte bands continue to gain in width with increase in migration distance.
Spatial temperature gradient has been suggested to also result in superior resolution (Rubey, W. A., J. High Res. Chromatogr. 1991, Vol. 14, p 542) compared to conventional gas chromatography. However, theoretical studies suggest that under ideal chromatographic conditions, that might not be the case (Ohline, R. W. and DeFord D. D., Anal. Chem. 1963, Vol. 35, pp 227-234). Regardless, TGGC is reported to be effective for improving loss of resolution or speed of analysis resulting from non-ideal chromatographic conditions such as poor sample introduction, column overloading, adsorption, and dead volume (Blumberg, L. M., Anal. Chem. 1992, Vol. 64, p. 2459; Blumberg, L. M., J. Chromatogr. Sci. 1997, Vo. 35, p. 451).
In liquid chromatography temperature manipulation has been used largely to heat or cool the entire column to a desired temperature for achieving improvement in chromatographic separation. For example, higher temperatures, which decreases viscosity of the mobile phase, leads to lower back pressures, making it possible to apply higher flow rates for achieving reduction in analysis time.
Temperature manipulation has also been used in conjunction with stationary phase conjugated to a temperature-sensitive polymer or copolymer. A stationary phase modified with a temperature sensitive polymer/copolymer binds analytes in a temperature dependent manner. For example, Muller et al. (J. Chromatogr. A, 1285 (2013) 97-109) describes a system for temperature-controlled fast protein liquid chromatography using a temperature-sensitive copolymer conjugated to the stationary phase in which an analyte is adsorbed at 42° C. and desorbed using a traveling cooling zone (at 22° C.) to obtain concentrated elution peaks.
Temperature manipulation in the form of a spatial temperature gradients may also, in theory, be used in liquid chromatography for achieving focused analyte peaks. However, in general, while the column temperature in liquid chromatography affects analyte retention in a manner similar to that observed in gas chromatography, the magnitude of the effect is much reduced, limiting the applicability of this technique to liquid or CO2-based chromatography. U.S. Pat. No. 8,226,825 describes a liquid chromatography method involving a spatial temperature gradient to achieve equilibrium gradient focusing. The gradient is applied in the form a temperature wave that moves repeatedly through a system of two or more chromatography columns (i.e., a non-fixed dynamic gradient). Analytes accumulate at select locations on the moving temperature wave and analyte peaks become narrower and more intense as the temperature wave is circulated about the system.